NMR

NMR

Application Note– Pulsar 002

Analysis of 60 MHz 1H Spectrum of Seed Oil

5 samples of 10 different seed oils were dissolved in chloroform

in a 1:1 ratio by volume. 1 mL of each sample was transferred

to 5 mm NMR tubes. A 60 MHz 1H NMR spectrum was acquired

for each of the samples. The area under the resonances A, C and

D was estimated by peak integration. The value of integral A was

set to 4 corresponding to 4 1H nuclei per glycerol backbone and

the other integrals were calculated as a ratio with respect to this

value. Providing estimates for the average number of olefinic (C)

and bis-allyllic 1H nuclei per triglyceride molecule. The results were

averaged across the five samples for each oil and are summarised

in table 1 along with the results for similar samples of pure

triglycerides, glyceryl tristearate, glyceryl trioleate and glyceryl

trilinoleate described previously.

Seed OilOlefinic

(1H per triglyceride)Bis-allyllic

(1H per triglyceride)

glyceryl tristearate (grey) - -

glyceryl trioleate (grey) 6.17 ± 0.07 -

glyceryl trilinoleate (grey) 12.1 ± 0.1 5.9 ± 0.2

Rapeseed (canola, blue) 8.4 ± 0.1 1.4 ± 0.1

Sunflower (black) 9.9 ± 0.1 2.6 ± 0.1

Olive (green) 6.08 ± 0.05 0.29 ± 0.04

Corn (maize, red) 9.9 ± 0.2 2.68 ± 0.09

Peanut (ground nut,purple)

7.0 ± 0.10.96 ± 0.04

Rice bran (orange) 7.5 ± 0.2 1.5 ± 0.2

Walnut (magenta) 11.5 ± 0.3 5.0 ± 0.2

Linseed (brown) 12.5 ± 0.2 6.6 ± 0.2

Coconut (cyan) 0.31 ± 0.05 -

Palm (pink) 3.58 ± 0.06 0.35 ± 0.02

Figure 7: Classification of seed oils using principal component analysis of the full spectrum.

The complete spectra can also be analysed using principal component

analysis (PCA). The principal components can then be used as

classifiers to distinguish between different oils. Figure 7 shows the

plot of the two most significant principal components and illustrated

the separation between the different oils. While it is convenient to

illustrate this classification with a two dimensional plot, statistically

more than two principal components can be used as discriminators.

Table 1: Average number of olefinic and bis-allyllic 1H nuclei estimated by peak integration. The colours in brackets after the name of the seed oil is the colour used to represent the data for each oil in figure 7.

Nuclear Magnetic Resonance (NMR) Spectroscopy applied to the analysis of Unsaturated Fat content in Seed Oils

Background

The constituents of plant derived oil fall under the general class of chemicals defined

as lipids. Prof. William Christie classifies them as “fatty acids and their derivatives,

and substances related biosynthetically or functionally to these compounds”. With

further clarification of the term fatty acid as “compounds synthesised in nature via

condensation of malonyl coenzyme A units by a fatty acid synthase complex. They

usually contain even numbers of carbon atoms in straight chains (commonly C14

to

C24

), and may be saturated or unsaturated; they can also contain other substituent

groups” [1]. See figure 1 below. Although a variety of lipids and starch comprise

the storage tissues of many food plants the major constituents of oil seeds are the triacylglycerol lipids; three fatty acid chains

attached as esters to a glycerol back bone. The fatty acid composition of oils is commercially important, e.g., high oleic acid

content, as found in olive oils, has been implicated in a reduced risk of heart disease and linoleic acid, found in sunflower oils, is

an essential nutritional fatty acid. High levels of these fatty acids can increase the commercial value of an oil whereas excessive

amount of linolenic acid can decrease the value since it is more susceptible to oxidation and problems with rancidity.

While most plant oils are produced for food and feed, up to 15% of some oils, e.g., soy and rapeseed, and 100% of certain

commodity oils, e.g., castor and tung, have non-food related industrial applications. Most oils comprise five primary fatty acids,

palmitic, stearic, oleic, linoleic and linolenic that can be utilised in the production of lubricants, surfactants, polymers and inks as

well as their used in food/feed applications. The fatty acids of commodity oils tend to have little or no nutrition value, however

their unusual chemical properties make them ideal for particular industrial applications. The most well known example is the

laurate fatty acid derived from coconut oil which has excellent foaming properties that makes it useful in the production of

anionic surfactants.

Application Note– Pulsar 002NMRNuclear Magnetic Resonance (NMR)

Spectroscopy applied to the analysis of Unsaturated Fat content in Seed Oils

Figure 1: Schematic representation of a typical triacylglycerol. R

1, R

2 and R

3 are

aliphatic chains that may or may not contain double bonds.

R2 C O CH

H2C O C

O

R1

H2C O C

O

R3

O

Glycerolipids

The class of substances described as lipids covers a broad

range of chemicals including fats, waxes, sterols and the

fat soluble vitamins whose primary functions include

energy storage, cell membrane structure and biochemical

signalling. Industrially the most common lipids are the

glycerolipids. The glycerolipids are mainly composed of

mono, di and tri-substituted glycerol (shown in figure

2). Of these the triacylglycerols, also referred to as

triglycerides, are by far the most common.

Triacylglycerols

In the triacylglycerols (TAG) each of the three hydroxyl

groups of the glycerol backbone are esterified with a fatty

acid. Typically the TAG will contain three different fatty

acids. There are many different types of TAG with their

level of saturation being dependent on the constituent

fatty acid chains. Figure 3 shows a triglyceride comprising

three different fatty acid chains; a saturated C16

chain fatty

acid (palmitic), a monounsaturated C18

chain (oleic) and a

polyunsaturated C18

chain fatty acid (linonelic). Different

seed oils have different levels of the fatty acid chains and

this composition determines their physical and chemical

properties, which in turn determines their commercial use.

Figure 2: Chemical structure of glycerol

H

C OHH1

CHO H2

CH OHH

3

H2C

O

HC O

O

O

O

H2C9 12 15

α

O

Nuclear Magnetic Resonance (NMR) Spectroscopy applied to the analysis of Unsaturated Fat content in Seed Oils

The different chemical groups give rise to specific signals in

an NMR spectrum, which at high field, typically 9.4 T, provide

a characteristic fingerprint or fatty acid profile that can be

used to identify an oil [2]. In this report it is demonstrated that

different chemical groups are still clearly identifiable in the 1H

NMR spectrum even at lower field, i.e., 1.4 T corresponding to a

proton Larmor frequency of 60 MHz.

Figure 3: Example of a TAG, with glycerol backbone on the right and from top to bottom the fatty acid chains, palmitic, oleic and α-linolenic.

NMR

Analysis

Anatomy of the 60 MHz, 1H Spectrum of Triacylglycerols

The 1H spectrum of glyceryl trilineolate (figure 4), a triglyceride where all the fatty acid chains derive from linoleic acid, is shown

in figure 5. It is possible to identify various resonances with specific chemical groups in the molecule. Resonance A originates

from the four 1H nuclei attached to carbon 1 and 3 on the glycerol backbone. This is a particularly useful signal because it is a

measure of the number of glyceride molecules in the sample and as such can be used as a reference signal. The signal from the

fifth 1H nuclei lies at about 5.3 ppm, directly below signal C. This must be taken into account when integrating the peaks.

Resonance C can be assigned to the 1H nuclei attached to carbons with a double bond, usually referred to as olefinic. This

peak is a measure of the total level of unsaturation in a triglyceride, regardless of whether it is in a monounsaturated or

polyunsaturated chain. Resonance D originates from a methylene group (-CH2-) sandwiched between two double bonds,

referred to chemically as bis-allyllic. This is specifically a measure of polyunsaturation.

Resonance E can be assigned to the terminal methyl group (-CH3) for each chain. Resonance F comes from the methylene group

on the fatty acid chain closest to the ester coupling with the glycerol back bone and resonance G comes from a methylene

group with a neighbouring double bond, often referred to chemically as an allylic group.

The large peak at approximately 1.2 ppm originates from the remaining methylene groups in the fatty acid chains. The integral

of resonances A, C and D can be used to quantify the level of unsaturation in a seed oil. The ratio of integral C and A gives

the average number of olefinic 1H nuclei per triglyceride and as such is a measure of overall unsaturated character. The ratio of

integral D and A provides an estimate of the number of bis-allyllic 1H nuclei, which is a measure of polyunsaturation

A A

BO O

CC

C DG

G E

O OO O

F

GC

DGCE

EG

CDGFF

NMR

Figure 4: Chemical structure of glyceryl trilinoleate

Figure 5: 1H spectrum of glyceryl trilinoleate obtained at 60 MHz.

C: -CH = CH-

A: Glyceride

D: =CH - CH2 - CH=

F: -00C-CH2-

G: =CH - CH2-

E: - CH3

To illustrate these estimates the

spectra of glyceryl trilinoleate

(bottom), glyceryl trioleate (middle)

and glyceryl tristearate (top) are

shown in figure 6. As shown in

figure 4 glyceryl trilinoleate is

comprised of a glyceryl backbone

with three linoleic fatty acid chains.

The spectrum shows both a large

olefinic resonance and a bis-allyllic

resonance.

Glyceryl trioleate comprises a glycerol

backbone with three oleic acid

chains. Oleic acid has a single double

bond in each chain and as can be

seen from the spectrum, there is a

significant olefinic resonances, but

no bis-allyllic resonance. The allyllic

resonance is clear in the spectrum of

both glyceryl trilinoleate and glyceryl

trioleate.

Figure 6: Comparison of the 60MHz 1H spectrum of glyceryl tristearate, glyceryl trioleate and glyceryl trilinoleate

Glyceryl tristearate comprises a

glycerol backbone with three stearic

acid chains. Stearic acid is a fully

saturated C18

chain and, as can be

seen from the spectrum there is no

bis-allyllic signal, no allyllic signal.

While there appears to be a rather

small olefinic resonance at ~5.3 ppm,

this is in fact the signal from the 1H

nuclei attached to carbon 2 on the

glycerol backbone.

The integral ratios are shown in

table 1 and it can be seen that

these are commensurate with

the unsaturated character of

the molecule

Glyceryl Tristearate

Glyceryl Trioleate

Glyceryl Trilinoleate

NMR

NMRApplication Note– Pulsar 002

visit www.oxford-instruments.com for more information

Summary

In conclusion, 60MHz NMR spectroscopy provides a suitable tool for

the study of seed oils. The chemical specificity of the spectral peaks

can be used as markers to estimate the levels different types of the

fatty acid chains. The analysis of the spectra could be extended

using a line fitting package. This could be used to estimate signal

amplitudes from overlapping resonances, especially those associated

with the methylene and methyl groups. The integral of this region of

the spectrum provides information about the average fatty acid chain

length.

The analysis of the whole spectrum using chemometric methods

such as principal component analysis provides a suitable framework

for classification of different types of oil. This work could be extended

beyond simple classification to provide a method for the detection of

adulterants or monitoring seasonal changes in oil crops.

[1] http://lipidlibrary.aocs.org/Lipids/whatlip/index.htm

[2] Guillen M. D. and Ruiz A. Eur. J. Lipid Sci Technol.

105 (2003) 501-507

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NMR